Summary How (and if) pain signaling is different in the central nervous system (CNS) compared to the rest of the body remains an open question. Nervous system inflammation and injury often lead to neuropathic pain (NP) where sensitivity of stimuli is greatly exaggerated or harmless stimuli are perceived as painful. Many patients suffering from these conditions do not respond to the standard treatment paradigms that are currently in use. This is primarily due to a lack of comprehensive understanding of the molecular mechanisms underlying transmission and processing of pain that limits treatment options. The pro-inflammatory cytokine, Interleukin-1? (IL-1) is known to facilitate pain sensitivity in various inflammatory neuropathic conditions in animal models of pain. IL-1 is capable of inducing multiple intracellular signaling cascades that ultimately leads to the expression of multiple inflammatory genes and sensation of pain. IL-1 signaling initiates when IL-1 binds to its cell surface receptor (IL-1R1) and its accessory protein (AcP). Recently a new accessory protein to IL-1 receptor (AcPb) has been identified that is only expressed in central nervous system (CNS). AcPb attenuates some, but not all, events in the IL-1 signaling pathway. This proposal aims to test a novel CNS-specific mechanism for regulation of IL-1 induced neuropathic pain signaling that is, hypothetically, regulated via clustering stoichiometry of IL-1 receptor and its two accessory proteins. We propose to experimentally verify existence of this regulatory mechanism. IL1R1, AcP and AcPb tagged with fluorescent proteins will be cloned and expressed in a model cell line and primary neurons for these experiments. We will employ advanced microscopic live cell measurements technologies, such as, Frster resonance energy transfer (FRET) to measure average distance (and molecular interaction) between two proteins, fluorescence correlation spectroscopy (FCS) to measure protein mobility (and association), and photon counting histogram (PCH) analysis to measure molecular brightness (and aggregation number) of the protein clusters. Additionally, calcium signaling and both mRNA and protein levels of cyclooxygenase-2 (COX- 2) and substance-P (Sub-P) will be measured to study pain-related functional aspects of IL-1 signaling. The long term goal of this research is to reconstruct multiple protein-protein interaction steps involved in IL-1R signaling pathway associated with imflammatory NP in live cells. Inflammatory neuropathic pain is a debelitating condition affecting many lives. A detailed molecular mechanism describing IL-1 in pain processing and perception, and how it is different in the CNS, may eventually lead to discovery of new drug targets for designing therapeutics for treatment options that are nonexistent today.